The innovation of the eukaryote cytoskeleton enabled phagocytosis, intracellular transport and cytokinesis, and is responsible for the diversity of eukaryotic morphologies. Still, the relationship between phenotypic innovations in the cytoskeleton and their underlying genotype is poorly understood. To explore the genetic mechanism of morphological evolution of the eukaryotic cytoskeleton we provide the first single cell transcriptomes from uncultured, free-living unicellular eukaryotes: the polycystine radiolarian Lithomelissa setosa (Nassellaria) and Sticholonche zanclea (Taxopodida). A phylogenomic approach using 255 genes finds Radiolaria and Foraminifera as separate monophyletic groups (together as Retaria), while Cercozoa is shown to be paraphyletic. Analysis of the genetic components of the cytoskeleton and mapping of the evolution of these to the revised phylogeny of Rhizaria reveal lineage-specific gene duplications and neofunctionalization of α and β tubulin in Retaria, actin in Retaria and Endomyxa, and Arp2/3 complex genes in Chlorarachniophyta. We show how genetic innovations have shaped cytoskeletal structures in Rhizaria, and how single cell transcriptomics can be applied for resolving deep phylogenies and studying gene evolution in uncultured protist species.

Long non-coding RNAs (lncRNAs) play important regulatory roles during animal development, and it has been hypothesized that an RNA-based gene regulation was important for the evolution of developmental complexity in animals. However, most studies of lncRNA gene regulation have been performed using model animal species, and very little is known about this type of gene regulation in non-bilaterians. We have therefore analysed RNA-Seq data derived from a comprehensive set of embryogenesis stages in the calcareous sponge Sycon ciliatum and identified hundreds of developmentally expressed intergenic lncRNAs (lincRNAs) in this species. In situ hybridization of selected lincRNAs revealed dynamic spatial and temporal expression during embryonic development. More than 600 lincRNAs constitute integral parts of differentially expressed gene modules, which also contain known developmental regulatory genes, e.g. transcription factors and signalling molecules. This study provides insights into the non-coding gene repertoire of one of the earliest evolved animal lineages, and suggests that RNA-based gene regulation was likely present in the last common ancestor of animals.

Recently, phylogenomic analyses have been used to assign the vast majority of eukaryotes into only a handful of supergroups. However, a few enigmatic lineages still do not fit into this simple picture. Such lineages may have originated early in the history of eukaryotes and are therefore of key importance in deduction of cellular evolution. In this study, we focus on two deeply diverging lineages, Diphyllatea and Thecamonadea. They are classified in the same phylum, Sulcozoa, but previous multigene phylogenetic analyses have included only one of these two lineages. It is therefore unclear whether they constitute one group or two distinct lineages. The study of rare genomic changes reveals that both have the fused dihydrofolate reductase (DHFR) and thymidylate synthase (TS) genes (i.e. DHFR–TS), which are separated in all other unikonts that have been investigated, indicating a possible close relationship. Their phylogenetic positions have implications for the classification of Sulcozoa and the early eukaryote evolution. Here we present a phylogenomic analysis of these species that include Illumina and 454 transcriptome data from two Collodictyon strains. A total of 42 mitochondrial proteins, which correspond to orthologs published from Thecamonas trahens (Thecamonadea), were used to reconstruct their phylogenies. In the resulting trees, Collodictyon appears as sister to Amoebozoa, whereas Thecamonas branches as the closest relative of Opisthokonta (i.e. the animal, fungi and unicellular Choanozoa). In contrast, the position of another early diverging eukaryote, Malawimonas, is unresolved. The separation of Collodictyon and Thecamonas in our studies suggests that the recently proposed Sulcozoa group is most likely paraphyletic. Furthermore, the data support the hypothesis that the two supergroups Opisthokonta and Amoebozoa, which comprise a great diversity of eukaryotes, have originated from a sulcozoan ancestor.

We have isolated cells of unculturable radiolarians from marine coastal waters. Individual cells were subjected to single cell whole genome amplification (SCWGA) and gene-targeted PCR. Using this approach we recover a surprisingly large diversity of sequences related to the enigmatic marine alveolate groups 1 and 2 (MALV I and MALV II) that most likely represent intracellular symbionts or parasites of the radiolarian cells. 18S rDNA phylogeny of the MALV sequences reveals 4 distinct clades of radiolarian associates here named Radiolarian Associated Sequences (RAS) 1-4. One clade of both phaeodarian and radiolarian associates and one clade of only phaeodarian associates are also identified. The MALV sequences cluster according to host type, i.e. sequences from associates identified in radiolarians, fish, copepods, ciliates or dinoflagellates are not intermixed but separated into distinct clades. This implies several independent colonizations of host lineages and links a large diversity of MALV to radiolarian-associated species. This demonstrates that radiolarians may be an important reservoir for MALV, making them a key group for understanding the impact of intracellular symbionts on the marine ecosystem. This study shows that applying SCWGA on unculturable cells is a promising approach to study the vast diversity and interactions of intracellular eukaryote organisms.

Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis.

Background:The implementation of high throughput sequencing for exploring biodiversity poses high demands on bioinformatics applications for automated data processing. Here we introduce CLOTU, an online and open access pipeline for processing 454 amplicon reads. CLOTU has been constructed to be highly user-friendly and flexible, since different types of analyses are needed for different datasets. Results:In CLOTU, the user can filter out low quality sequences, trim tags, primers, adaptors, perform clustering of sequence reads, and run BLAST against NCBInr or a customized database in a high performance computing environment. The resulting data may be browsed in a user-friendly manner and easily forwarded to downstream analyses. Although CLOTU is specifically designed for analyzing 454 amplicon reads, other types of DNA sequence data can also be processed. A fungal ITS sequence dataset generated by 454 sequencing of environmental samples is used to demonstrate the utility of CLOTU. Conclusions:CLOTU is a flexible and easy to use bioinformatics pipeline that includes different options for filtering, trimming, clustering and taxonomic annotation of high throughput sequence reads. Some of these options are not included in comparable pipelines. CLOTU is implemented in a Linux computer cluster and is freely accessible to academic users through the Bioportal web-based bioinformatics service (http://www.bioportal.uio.no).

Background: Large multigene sequence alignments have over recent years been increasingly employed for phylogenomic reconstruction of the eukaryote tree of life. Such supermatrices of sequence data are preferred over single gene alignments as they contain vastly more information about ancient sequence characteristics, and are thus more suitable for resolving deeply diverging relationships. However, as alignments are expanded, increasingly numbers of sites with misleading phylogenetic information are also added. Therefore, a major goal in phylogenomic analyses is to maximize the ratio of information to noise; this can be achieved by the reduction of fast evolving sites. Results: Here we present a batch-oriented web-based program package, named AIR that allows 1) transformation of several single genes to one multigene alignment, 2) identification of evolutionary rates in multigene alignments and 3) removal of fast evolving sites. These three processes can be done with the programs AIR-Appender, AIR-Identifier, and AIR-Remover (AIR), which can be used independently or in a semi-automated pipeline. AIR produces user-friendly output files with filtered and non-filtered alignments where residues are colored according to their evolutionary rates. Other bioinformatics applications linked to the AIR package are available at the Bioportal (www.bioportal.uio.no), University of Oslo; together these greatly improve the flexibility, efficiency and quality of phylogenomic analyses. Conclusions: The AIR program package allows for efficient creation of multigene alignments and better assessment of evolutionary rates in sequence alignments. Removing fast evolving sites with the AIR programs has been employed in several recent phylogenomic analyses resulting in improved phylogenetic resolution and increased statistical support for branching patterns among the early diverging eukaryotes.

Cordyceps sinensis is a reputed medicinal fungus growing parasitically on buried larvae of ghost moths in Asian high-altitude grassland ecosystems. We have analysed the intraspecific ITS nrDNA (ITS1, 5.8S gene, ITS2) variation among 71 sequences of C. sinensis available in EMBL/Genbank. The ITS sequences, submitted to Bayesian ML analyses, were distributed into five groups, referred to as A-E. Nine of the sequences (groups D and E) grouped with distantly related hypocrealean/clavicipitalean taxa and are interpreted as sequences erroneously accessioned under wrong taxon names. The remaining 62 sequences constituted three highly supported clades (groups A-C), that may represent cryptic (phylogenetic) species currently ascribed to C. sinensis. A remarkably high sequence divergence occurred in the 5.8S gene between the three groups. Sequences of groups B and C showed accelerated substitution rates and high AT nucleotide bias. We hypothesize that the accelerated evolution and AT bias have been caused by a shift in life historical attributes or ecology. We also suggest that the recorded differences in medicinal effects among C. sinensis populations may be attributed to the existence of genetically differentiated chemotypes in this morphotaxon.

Serpula himantioides (Boletales, Basidiomycota) produces thin resupinate basidiocarps on dead coniferous wood worldwide and causes damage in buildings as well. In this study, we present evidence for the existence of at least three phylogenetically defined cryptic species (referred to as Sib I–III) within the morphospecies S. himantioides, a conclusion based on analyses of sequence data from four DNA regions and amplified fragment length polymorphisms (AFLPS). A low degree of shared sequence polymorphisms was observed among the three lineages indicating a long-lasting separation. The AFLPs revealed two additional subgroups within Sib III. Results from mating studies were consistent with the molecular data. In Sib III, no correspondence between genetic and geographical distance was observed among isolates worldwide, presumably reflecting recent dispersal events. Our results indicate that at least two of the lineages (Sib II and Sib III) have wide sympatric distributions. A population genetic analysis of Sib III isolates, scoring sequence polymorphisms as codominant SNP markers, indicates that panmictic conditions exist in the Sib III group. This study supports the view that cryptic speciation is a common phenomenon in basidiomycete fungi and that phylogenetic species recognition can be a powerful inference to detect cryptic species. Furthermore, this study shows that AFLP data are a valuable supplement to DNA sequence data in that they may detect a finer level of genetic variation.

The basidiomycete genus Galerina Earle accommodates more than 300 small brown-spored agarics worldwide, predominantly described from the Northern hemisphere. The delimitation of species and infrageneric units hitherto has been based on morphological and, to some extent, ecological characters. In this study we have analyzed nuclear ribosomal LSU and ITS sequences to reveal infrageneric phylogeny and the phylogenetic placement of Galerina among the dark-spored agarics. Sequences from 36 northern hemisphere Galerina species and 19 other dark-spored taxa were analyzed, some of them obtained from EMBL/GenBank. Our results, received from Bayesian and distance methods, strongly suggest that Galerina is a polyphyletic genus. The LSU analysis shows that Galerina is composed of three or four separate monophyletic main groups. In addition, a few species cluster together with other dark-spored agarics. The same groups are recognized in the ITS tree and they correspond roughly to previously recognized subgenera or sections in Galerina. With high support our LSU analysis suggests that Gymnopilus is a monophyletic genus and that Gymnopilus and one of the Galerina lineages ("mycenopsis") are sister groups. The analyses further indicate that the Galerina lineages, as well as the genus Gymnopilus, could be referred to a strongly emendated family Strophariaceae, which corresponds largely to the family as circumscribed by Kühner (1980). Our results affirm that morphological characters often are highly homoplastic in the agarics. At the present stage formal taxonomic consequences or nomenclatural changes are not proposed.

During the last decade, the improvement of high-throughput sequencing (HTS) techniques has substantially increased our insight into microbial ecology, by capturing a much more detailed picture of their diversity. Due to the increasing amount of data obtained by HTS methods, specific questions on the biogeography and factors explaining the distribution of these microorganisms can now be fully examined. Here, the composition and structure of a metabolically active benthic protistan community was investigated along a salinity gradient of closely associated marine and freshwater systems. Amplicons of rDNA and rRNA, were sequenced using the Illumina MiSeq technology. This approach seemed to successfully capture the majority of the protist community in these habitats. The marine environments displayed a higher species richness compared to the lacustrine systems investigated, and a considerable difference in protist composition was observed between marine and freshwater systems. Nevertheless, some protists were found in both saline and freshwater environments, indicating the ability to survive rather diverse environmental conditions. It is noteworthy that some of the OTUs from both habitats form putative new clades in the enigmatic X-cell group.

Liang, Zhe; Olsen, Odd-Arne; Shalchian-Tabrizi, Kamran; Wilson, Robert & Johansen, Wenche (2012). The DEK1 protein evolved from an ancient form of membrane bound calpain and is highly conserved in land plants.

Demko, Viktor; Perroud, Pierre-Francois; Johansen, Wenche; Wilson, Robert Charles; Liang, Zhe; Shalchian-Tabrizi, Kamran & Olsen, Odd-Arne (2011). The DEK1 and CRINKLY4 proteins play a fundamental role in the development of land plants - from Arabidopsis back to the moss Physcomitrella patens.

Pseudochattonella farcimen (Eikrem, Edvardsen, et Throndsen) is an ichthyotoxic alga within the Dictyochophyceae (Heterokonta), which has been shown to form blooms in Scandinavian waters every year since 1998. To improve our understanding of the biology of this alga and to facilitate future genomic studies, we report the sequencing and analysis of >10,000 expressed sequence tags (ESTs) corresponding to 8149 unique sequences from this species. A direct comparison with EST libraries from other heterokonts revealed several functional categories to be significantly overrepresented among the P. facimen ESTs, such as genes involved in cell communication, transporters, or genes targeted to cell organelles. Interestingly, P. farcimen ESTs also code for a high proportion (1.4%) of proteins related to fatty acid metabolism, including eight fatty acid desaturases and two phospholipase A2 genes. Three of the desaturases belong to a family of delta-4 desaturases, known so far only from haptophytes, where they catalyze the conversion of n3-docosapentaenoic (n3-DPA) acid to docosahexaenoic acid (DHA). These findings may partially explain the unusual fatty acid profiles observed in P. farcimen and are discussed both from an evolutionary point of view and in relation the ichthyotoxic effects of this alga.

Ministeria vibrans is a unicellular protozoan belonging to the phylum Choanozoa, the evolutionary closest related group to the animals. Ministeria is a minute bacteria-eating cell possessing slender radiating tentacles, that has recently been shown to be the most close relative to Capsaspora, another protozoan with thread-like (filose) tentacles. Forming the novel clade Filasterea, Ministeria/Capsaspora was shown to be sister to animals and choanoflagellates. Presumably, their last common ancestor evolved filose tentacles well before they were aggregated as a periciliary collar in the choanoflagellates. Several successive evolutionary innovations occurred among the unicellular closest relatives of the animals prior to the origin of multicellularity. We have analysed an EST library from this early branching choanozoan organism and have found many domains of key significance for animal cell biology. New domain combinations as well as domain duplications must have taken place along the path to multicellularity.

Cordyceps sinensis (Berk.) Sacc. grows parasitically on buried larvae of ghost moths (Thitarodes/Hepialus spp.) in Asian high-altitude grasslands and is known as a highly reputed medicinal fungus. In this study, the intraspecific variation in C. sinensis was analyzed employing 71 nrDNA ITS sequences accessioned in GenBank. Bayesian maximum likelihood analyses distributed the C. sinensis ITS sequences into five groups, referred to as A-E. Two groups (D-E), including a total of nine C. sinensis sequences as well as other Hypocreales/Clavicipitales taxa, were interpreted as erroneously accessioned sequences. The remaining sequences (62) constituted three highly supported clades (groups A, B and C), that may represent cryptic (phylogenetic) species currently ascribed to C. sinensis. Two sequences, accessioned as C. multiaxialis and C. nepalensis, also merged with the C. sinensis sequences of clades A-C. Sequences of groups B and C showed accelerated substitution rates and high AT nucleotide bias throughout the entire ITS region. A remarkably high sequence variation occurred in the 5.8S gene, which far exceeds what is normally observed in fungi. Compelling evidence from other studies exists for that reduced recombination frequencies can lead to AT bias due to lack of GC biased gene conversion, a process coupled to the meiosis. We hypothesize that a transition in life history attributes or ecology has happened among the putative cryptic species of C. sinensis, leading to accelerated evolution and AT bias. We also suggest that observed differences in medicinal effects among C. sinensis populations may be attributed to the existence of cryptic species in this morphotaxon.

The basidiomycete genus Galerina Earle accommodates more than 300 small brownspored agarics world wide, predominantly described from the Northern hemisphere. The delimitation of species and infrageneric units has hitherto been based on morphological and, to some extent, ecological characters. In this study, we have analysed nuclear ribosomal LSU and ITS sequences in order to reveal infrageneric phylogeny and the phylogenetic placement of Galerina among the dark-spored agarics. Sequences from 36 Northern hemisphere Galerina species and 19 other dark-spored taxa were analysed, some of them obtained from EMBL/GenBank. Our results, received from Bayesian and distance methods, strongly suggest that Galerina is a polyphyletic genus. The LSU analysis shows that Galerina is composed of three or four separate monophyletic main groups. In addition, a few species cluster together with other dark-spored agarics. The same groups are recognized in the ITS tree and they correspond roughly to previously recognized subgenera or sections in Galerina. With high support, our LSU analysis suggests that Gymnopilus is a monophyletic genus and that Gymnopilus and one of the Galerina lineages (�mycenopsis�) are sister groups. The analyses further indicate that the Galerina lineages, as well as the genus Gymnopilus, could be referred to a strongly emendated family Strophariaceae which corresponds largely to the family as circumscribed by Kühner (1980). Our results affirm that morphological characters often are highly homoplastic in the agarics. At the present stage formal taxonomic consequences or nomenclatural changes are not proposed.

Since 1998 a heterokont flagellate, initially identified as Chattonella aff. verruculosa, has formed recurrent extensive blooms in the North Sea and Skagerrak causing mortality in farmed salmon. Strains were isolated from a 2001 bloom off the south coast of Norway and compared with a �Chattonella verruculosa� strain from Japan. The cells in Norwegian strains are oblong to round with numerous chloroplasts and mycocyst-like organelles. The complete SSU and partial LSU rRNA gene sequences from strains from Norway and Japan were similar, but not identical, and differed clearly from rDNA sequences of other Raphidophytes. Phylogenetic trees were constructed and their relationships to other heterokontophytes determined. C. aff. verruculosa and the Japanese �C. verruculosa� appeared in a clade with members of Dictyochophyceae. Ultrastructure (e.g. the structure of flagella and flagellar apparatus, position of nucleus), morphology and pigment data (chlorophylls a, c1, c2, c3, fucoxanthin, diadinoxanthin and 19�butanoyloxyfucoxanthin) support this affinity. A new name and classification will be proposed.